Blood cells, including erythrocytes, lymphocytes, neutrophils, and monocytes, as well as non-cellular components such as platelets, serve specialized life-sustaining roles. It follows, then, that a method that can evaluate drugs, environmental factors, radiation exposures, or other agents for their ability to adversely affect stem cells, fully differentiated blood cells, or any intermediate stage cell, would be extremely useful for hazard identification purposes. This is one important application of the present invention.
Large-scale accidental, terrorist, or warfare-related radiation exposure scenarios will severely challenge the nation's medical community. The most effective allocation of resources will be required, especially in the hours and days immediately following a disaster, when many critical treatment decisions must be made. Thus, in order to deal with large-scale radiological events, there is a need for assays that supply information regarding dose distribution to tissue compartments of greatest clinical significance. The present invention addresses this need by representing a high throughput system that can be used to rapidly estimate radiation dose based on perturbations to the hematopoietic system. As described herein, these measurements can be accomplished with microliter quantities of blood, with minimal processing steps, and with currently available instrumentation.
One well known radiation biodosimetry assay is based on the kinetics by which lymphocytes are depleted from peripheral blood circulation following exposure to radiation (Goans et al., Health Phys. 81, 446-449 (2001)).
The kinetics of depletion is considered more useful than a single measurement, since the latter does not provide a very specific indicator of radiation exposure due to the amount of inter-individual variation that exists in the human population.
Depletion of lymphocytes from circulation, as well as many other significant manifestations of toxicity, can often be attributed to programmed cell death, or apoptosis. An early event in the apoptotic program is loss of mitochondrial membrane potential. Some fluorescent dyes accumulate in mitochondria in proportion to their membrane potential. Thus, decreased or altered fluorescent intensity denotes mitochondrial dysfunction, and these staining characteristics have been widely used to detect cells undergoing apoptosis. Relevant to the invention disclosed herein, it has been observed that among several apoptosis labeling techniques, assessing loss of mitochondrial membrane potential is particularly advantageous in regard to detecting blood lymphocytes that have been damaged by ionizing radiation (Benderitter et al., Radiation Res., 158, 464-474 (2002)). These authors speculate that elimination of dying lymphocytes by phagocytes limits the value of other methods that detect apoptotic cells based on features that occur downstream of mitochondrial perturbations. Even so, the blood processing techniques employed by these investigators is less than ideal for radiation biodosimetry purposes in at least two respects. Firstly, Benderitter et al. isolated blood lymphocytes on a Ficoll-Histopaque gradient, and subsequently washed the cells two times before accomplishing quantitative analysis. These extra processing steps require time, labor, reagents, and equipment that the invention described herein does not. Secondly, these processing steps have the potential to introduce errors in terms of cell enumeration, as well as assessments of cell health.
It would be desirable, therefore, to provide an analysis of markers that enhance the value of both single and dual time-point lymphocyte counts for the purpose of radiation dose estimation. The present invention is directed to overcoming this deficiency of the prior art.